Eriodictyol {Figure 1; [(S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4-one]} was prepared at the USDA-ARS Horticultural Research Laboratory (Fort Pierce, Florida, USA). A stock solution was prepared in dimethylsulfoxide (DMSO) at a concentration of 1 M and was kept at 4°C in the dark. The final concentration of DMSO in the assays described below was ≤ 0.1% and had no effects on gingival keratinocytes and macrophages.

Porphyromonas gingivalis ATCC 33277 was cultivated in an anaerobic chamber (80% N₂, 10% CO₂, 10% H₂) at 37°C in Todd-Hewitt broth (THB; Becton Dickinson and Company, Sparks, MD, USA) containing 0.001% (w/v) hemin and 0.0001% (w/v) vitamin K.

The previously characterized B11 human gingival keratinocyte cell line [25] was cultivated in keratinocyte serum-free medium (K-SFM; Life Technologies Inc., Burlington, ON, Canada) supplemented with 50 μg/mL of bovine pituitary extract, 5 ng/mL of human epidermal growth factor, 100 μg/mL of penicillin G-streptomycin, and 0.5 μg/mL of amphotericin B. Gingival keratinocytes (3 × 10⁵ cells/well) were seeded in black wall, clear flat bottom 96-well microplates (Greiner Bio-One North America, Monroe, NC, USA) and were incubated overnight at 37°C in a 5% CO₂ atmosphere. ROS production was measured using a fluorometric assay to monitor the oxidation of 2′,7′-dichlorofluorescein-diacetate (DCF-DA; Sigma-Aldrich Canada Co., Oakville, ON, Canada) into a fluorescent compound. DCF-DA was freshly prepared in DMSO at a concentration of 40 mM. The cells were washed with Hank's balanced salt solution (HBSS; HyClone Laboratories, Logan, UT, USA) and were incubated for a further 30 min in the presence of 100 μM DCF-DA in HBSS. Excess DCF-DA was discarded, and the keratinocytes were washed with HBSS. The cells were then treated with P. gingivalis cells at a multiplicity of infection (MOI) of 100 either in the presence or absence of hydrogen peroxide (H₂O₂; 1 mM) and in the absence or presence of eriodictyol (40, 80, or 160 μM) in HBSS. H₂O₂ was used as an inducer of oxidative stress [26, 27]. The fluorescence emission indicating ROS production was then recorded every 20 min for 3 h at 37°C using a Synergy 2 microplate reader (BioTek Instruments, Winooski, VT, USA) with a 485 nm excitation filter and a 528 nm emission filter. Assays were carried out in triplicate in three independent experiments, and the means ± standard deviations were calculated.

To assess the effect of eriodictyol on the adhesion of P. gingivalis to gingival keratinocytes (B11 cell line), the bacteria were first labeled with fluorescein isothiocyanate (FITC). Briefly, a 24-h culture (10 mL) was harvested by centrifugation (9,000 × g for 5 min), washed once in 10 mm phosphate-buffered saline (PBS; pH 7.2), and suspended in 0.5 m sodium bicarbonate buffer (pH 8) containing 0.03 mg/mL of FITC. The bacterial suspension was incubated in the dark at 37°C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (9,000 × g for 5 min) and were suspended in 10 mL of K-SFM. Gingival keratinocytes cultured as described above were seeded at 1 × 10⁶ cells/mL in sterile black wall, clear flat bottom microplates (Greiner Bio-One North America Inc.) and were incubated overnight at 37°C in a 5% CO₂ atmosphere. The medium was then removed by aspiration, and the confluent cell monolayers were pre-incubated with eriodictyol (40, 80, or 160 μM; in K-SFM) for 30 min at 37°C in a 5% CO₂ atmosphere. FITC-labeled P. gingivalis was added at an MOI of 1,000, and the monolayers were incubated for a further 4 h at 37°C in a 5% CO₂ atmosphere. Un-adhered bacteria were then removed, and the wells were washed twice with PBS. Relative fluorescence units (RFU) corresponding to the level of adhered bacteria were determined using a Synergy 2 microplate reader (BioTek Instruments) at an excitation wavelength of 495 nm and an emission wavelength of 525 nm. Wells with no P. gingivalis were used as controls to measure basal auto-fluorescence. Control wells without eriodictyol were used to determine 100% adherence values. Assays were performed in triplicate in three independent experiments, and the means ± standard deviations were calculated.

U937 human monocytes (CRL-1593.2; American Type Culture Collection, Manassas, VA, USA) were cultivated in Roswell Park Memorial Institute 1640 medium (RPMI; Life Technologies Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 μg/mL of penicillin G/streptomycin at 37°C in a 5% CO₂ atmosphere. Prior to stimulation with P. gingivalis, the monocytes (1 × 10⁶ cells/mL) were differentiated into adherent macrophage-like cells by incubating them in RPMI-10% FBS containing 100 ng/mL of phorbol myristic acid (PMA; Sigma-Aldrich Canada Co.) for 48 h in 12-well microplates. After PMA treatment, the adherent macrophage-like cells were washed to remove remaining PMA and non-adherent cells. The differentiated cells were further incubated in RPMI-10% FBS without PMA for 24 h. Then, the adherent macrophage-like cells were maintained in RPMI-1% FBS and incubated overnight at 37°C in a 5% CO₂ atmosphere. The macrophage-like cells were pre-treated for 2 h with eriodictyol (80, 160, or 320 μM) in RPMI-1% FBS prior to stimulating them with P. gingivalis cells at an MOI of 100. After a 24-h incubation at 37°C in a 5% CO₂ atmosphere, the culture medium supernatants were collected and were kept at −20 °C until used. To exclude any effects resulting from a loss of cell viability, the cytotoxicity of eryodictyol at the concentrations used was assessed with an MTT (3-[4,5-diethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) colorimetric assay (Roche Diagnostics, Laval, QC, Canada) performed according to the manufacturer's protocol. Cells incubated in culture medium in the absence of eriodictyol and bacteria were used as controls. Enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA; Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA) were used to determine IL-1β, IL-6, IL-8, TNF-α, MMP-2, MMP-8, and MMP-9 concentrations according to the manufacturers' protocols. Assays were performed in triplicate in three independent experiments, and the means ± standard deviations were calculated.

The human monoblastic leukemia cell line U937 3xκ B-LUC, a subclone of the U937 cell line stably transfected with a luciferase gene linked to a promoter of three NF-κB-binding sites [28], was used to investigate the effect of eriodictyol on P. gingivalis-induced activation of the NF-κB signaling pathway. These cells were cultivated in RPMI-10% FBS, 100 μg/mL of penicillin G/streptomycin, and 75 μg/mL of hygromycin B at 37°C in a humidified incubator with a 5% CO₂ atmosphere. The U937 3xκB-LUC monocytes were seeded (10⁵ cells/well) in the wells of black wall, black bottom, 96-well microplates (Greiner Bio-One North America) and were pre-incubated with eriodictyol (80, 160, or 320 μM) for 30 min. The monocytes were then stimulated with P. gingivalis at an MOI of 100 for 6 h. Wells containing monocytes without bacteria or eriodictyol were used as controls. Bright-Glo reagent (Promega Corporation, Durham, NC, USA) was used according to the manufacturer's protocol to measure luciferase activity and determine NF-κB activation. Luminescence was monitored using a Synergy 2 microplate reader. Assays were carried out in triplicate in three independent experiments, and the means ± standard deviations were calculated.

The effect of eriodictyol (80, 160, and 320 μM) on MMP activity was assessed using an MMP-9 Inhibitor Screening Fluorometric Assay Kit (Abcam Inc., Toronto, ON, Canada) according to the manufacturer's protocol. MMP-9 activity was monitored by recording fluorescence with a 328 nm excitation filter and a 420 nm emission filter for 20 min. N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH; 1 mM) was used as a control inhibitor of MMP-9 activity. Assays were performed in triplicate in three independent experiments, and the means ± standard deviations were calculated.

To determine the effect of eriodictyol on the collagenase activity of P. gingivalis, a 48-h culture was centrifuged at 10,000 × g for 10 min. The culture supernatant was incubated at 37°C for 3 h with eriodictyol (80, 160, or 320 μM) and 100 μg/mL of fluorogenic DQ substrate collagen (Molecular Probes, Eugene, OR, USA). The fluorescence corresponding to collagen degradation was measured every 30 min using a Synergy 2 microplate reader with a 495 nm excitation filter and a 525 emission filter. Leupeptin (1 μM) was used as a positive inhibitor control. Assays were performed in triplicate in three independent experiments, and the means ± standard deviations were calculated.

A one-way ANOVA with a post-hoc Bonferroni multiple comparison test was used to analyze the data. Results were considered statistically significant at p < 0.05, p < 0.01, or p < 0.001.

The production of ROS by gingival keratinocytes may cause deleterious effects on the oral epithelium. It was first shown that P. gingivalis (MOI = 1,000) time-dependently increased the production of ROS by gingival keratinocytes. Following a 3-h contact, we observed a 4.3-fold increase in ROS compared to untreated control cells (Figure 2). However, in the presence of eriodictyol, at all the concentrations tested (40, 80, and 160 μM), P. gingivalis-induced ROS production was significantly attenuated, with a 24.0–30.6% reduction after the 3-h contact (Figure 2). The ability of eriodictyol to impair ROS production by gingival keratinocytes treated with P. gingivalis combined with H₂O₂, a well-known inducer of oxidative stress [26, 27], was also investigated. As reported in Figure 3, P. gingivalis and H₂O₂ acted synergistically to induce ROS production by gingival keratinocytes. More specifically, following a 3-h contact, the P. gingivalis + H₂O₂ combination induced a 19.5-fold higher production of ROS compared to the amount of ROS produced by cells treated with P. gingivalis or H₂O₂ alone. Eriodictyol attenuated ROS production, with a 160 μM concentration causing a 52.9% inhibition after a 3-h contact.

Analyses were then conducted to determine the ability of eriodictyol to inhibit the adhesion of P. gingivalis to gingival keratinocytes (B11 cell line) as a potential mechanism involved in the inhibition of P. gingivalis-induced ROS production. Eriodictyol dose-dependently reduced the adherence of FITC-labeled P. gingivalis to gingival keratinocytes (Figure 4). More specifically, in the presence of 160 μM eriodictyol, adherence was inhibited by 30.8%.

To investigate the anti-inflammatory effect of eriodictyol, macrophages were pretreated for 2 h with various concentrations (80, 160, or 320 μM) of this flavanone before being challenged with P. gingivalis cells at an MOI of 100. To exclude the possibility that the reduction in levels may have resulted from cell toxicity, we evaluated the viability of macrophages that had been treated for 24 h with eriodictyol. No obvious cytotoxic effects were detected with eriodictyol at a final concentration of 320 μM. Cell viability was ≥92% compared with the untreated control (data not shown). The secretion of IL-6, IL-8, TNF-α, and IL-1β increased significantly when the macrophages were stimulated with P. gingivalis compared to untreated cells (Figure 5). More specifically, the secretion of IL-6, IL-8, TNF-α, and IL-1β increased 17.4-, 8.5-, 138.5-, and 4.2-fold, respectively. Eriodictyol, at all the concentrations tested, significantly decreased the P. gingivalis-induced secretion of the four cytokines. In the presence of 160 μM eriodictyol, the secretion of IL-6, IL-8, TNF-α, and IL-1β decreased by 99.9, 70.8, 95.2, and 78.5%, respectively.

The same protocol was used to assess the effect of eriodictyol on MMP production by P. gingivalis-treated macrophages. The secretion of MMP-2, MMP-8, and MMP-9 increased significantly when the macrophages were challenged with P. gingivalis compared with untreated cells (Figure 6). Eriodictyol dose-dependently reduced the production of all three MMPs. At a concentration of 160 μM, eriodictyol reduced the P. gingivalis-induced production of MMP-2, MMP-8, and MMP-9 by 36.9, 99.9, and 82.5%, respectively.

An alternative model was used to assess the anti-inflammatory effect of eriodictyol. Since NF-κB is a transcription factor implicated in inflammatory processes leading to the production of cytokines and MMPs, we evaluated the effect of eriodictyol on NF-κB activation using the U937 3xκ B-LUC monocytic cell line. As shown in Figure 7, eriodictyol dose-dependently reduced P. gingivalis-modulated NF-κB activation. At a concentration of 160 μM, eriodictyol reduced NF-κB activation by 80.7%.

Eriodictyol was further studied for its ability to inhibit MMP activity. We used MMP-9 as a model and showed that eriodictyol inhibits the catalytic activity of MMP-9 in a dose- and time-dependent manner (Figure 8). After the 20-min incubation time, eriodictyol at 80, 160, and 320 μM inhibited MMP-9 activity by 19.9, 27.4, and 58.0%, respectively.

Given that P. gingivalis may act in concert with MMPs to cause the destruction of periodontal connective tissue, we assessed the effect of eriodictyol on the collagenase activity of this periodontal pathogen. We showed that eriodictyol inhibits the degradation of type I collagen by P. gingivalis in a dose- and time-dependent manner. After a 4-h incubation, the lowest concentration (80 μM) of eriodictyol inhibited collagen degradation by 9.6% while the highest concentration (320 μM) inhibited collagen degradation by 30.1% (Figure 9).